Generally, gel-electrophoretic methods with autoradiographical or optical detection are used for DNA and RNA sequence analysis, e.g. in disease diagnosis, toxicological test procedures, genetic research and development, as well as in the agrarian and pharmaceutical sectors.
To illustrate the most significant gel-electrophoretic method with optical detection (Sanger method), FIG. 1b shows a DNA fragment with primer. In the Sanger method, a DNA-containing solution is divided into four samples and the primer of each sample is covalently modified with a fluorescent dye that emits at a distinct wavelength. As illustrated in FIG. 1b, deoxyribonucleoside triphosphate of bases A (adenine), T (thymine), C (cytosine), and G (guanine), i.e. dATP, dTTP, dCTP, and dGTP, are added to each sample to enzymatically replicate the single strand, starting at the primer, by means of DNA polymerase 1. In addition to the four deoxyribonucleoside triphosphates, each reaction mixture also contains sufficient 2′,3′-dideoxy analog (FIG. 1a) of one of these nucleoside triphosphates as a blocking base (one of each of the four possible blocking bases per sample) to terminate replication at all possible binding sites. After combining the four samples, all lengths of replicated DNA fragments having blocking-base-specific fluorescence result and can be gel-electrophoretically sorted according to length and characterized using fluorescent spectroscopy (FIG. 1c).
Another optical detection method is based on the accumulation of fluorescent dyes such as e.g. ethidium bromide on oligonucleotides. The fluorescence of such dyes increases in comparison with the free solution of the dye by about 20-fold when they accumulate on double-stranded DNA or RNA and can therefore be used to detect hybridized DNA or RNA.
In radiolabeling, 32P is built into the phosphate skeleton of the oligonucleotides, with 32P usually being added to the 5′-hydroxyl end by means of polynucleotide kinase. Thereafter, the labeled DNA is preferably cleaved, under defined conditions, at one of each of the four nucleotide types, such that an average of one cleavage per chain results. Thus, for a given base type, there are present in the reaction mixture chains extending from the 32P-label to the position of that base (if there are multiple appearances of the base, chains of varying lengths will result accordingly). The four fragment mixtures are then gel-electrophoretically separated on four lanes. Thereafter, an autoradiogram of the gel is prepared, from which the sequence can be directly read.
Some years ago, a further method of DNA sequencing was developed on the basis of optical (or autoradiographical) detection, namely sequencing by means of oligomer hybridization (cf. e.g. Drmanac et al., Genomics 4, (1989), pp. 114–128 or Bains et al., Theor. Biol. 135, (1988), pp. 303–307). In this method, a complete set of short oligonucleotides, or oligomers (probe oligonucleotides), e.g. all 65,536 possible combinations of the bases A, T, C, and G of an oligonucleotide octamer are bound to a support. The attachment occurs in an ordered grid consisting of 65,536 test sites, with each larger amount of an oligonucleotide combination defining one test site, and the position of each individual test site (oligonucleotide combination) is known. On such a hybridization matrix, the oligomer chip, a DNA fragment whose sequence is to be determined, the target, is labeled with fluorescent dye (or 32P) and hybridized under conditions that allow only one specific double-strand formation. In this way, the target DNA fragment attaches only to the oligomers (in this example to the octamers) whose complementary sequence corresponds exactly to a portion (an octamer) of its own sequence. Thus, all of the oligomer sequences (octamer sequences) present in the fragment are determined by means of optical (or autoradiographical) detection of the binding position of the hybridized DNA fragment. Due to the overlapping of neighboring oligomer sequences, the continuous sequence of the DNA fragment can be determined using suitable mathematical algorithms. The advantages of this method lie in, among other things, the miniaturization of the sequencing and thus in the enormous amount of data that can be simultaneously captured in one operation. In addition, primer and gel-electrophoretic separation of the DNA fragments can be dispensed with. This principle is demonstrated by example in FIG. 2 for a 13-base-long DNA fragment.
The use of radioactive labels in DNA/RNA sequencing is associated with several disadvantages, such as e.g. elaborate, legally required safety precautions in dealing with radioactive materials, radiation, spatially limited resolution capacity (maximum 1 mm2) and sensitivity that is only high when the radiation of the radioactive fragments act on an X-ray film for an appropriately long time (hours to days). Although the spatial resolution can be increased by means of additional hardware and software, and the detection time can be decreased by means of β-scanners, both of these involve considerable additional costs.
Some of the fluorescent dyes that are commonly used to label the DNA (e.g. ethidium bromide) are mutagenic and require appropriate safety precautions, as does the use of autoradiography. In nearly every case, the use of optical detection requires the use of one or more laser systems, and thus experienced personnel and appropriate safety precautions. The actual detection of the fluorescence requires additional hardware such as e.g. optical components for amplification and, in the case of varying stimulation and query wavelengths as in the Sanger method, a control system. Thus, depending on the stimulation wavelengths required and the detection performance desired, considerable investment costs may result. In sequencing by means of hybridization on the oligomer chip, detection is even more costly because, in addition to the stimulation system, high-resolution CCD cameras (charge coupled device cameras) are needed for 2-dimensionally detecting the fluorescent spots.
Thus, although there are quantitative and extremely sensitive methods for DNA/RNA sequencing, these methods are time consuming, require elaborate sample preparation and expensive equipment, and are generally not available as portable systems.